2.1 Field of the Invention
The present invention relates generally to the field of analytical chemistry, and, more particularly, to the separation of components of complex mixtures. Still more particularly, the present invention relates to the separation of substances having defined isoelectric points from mixtures. The present invention has applications in the fields of biochemistry, proteomics, and clinical chemistry.
2.2 Background
Proteomics, the analysis of the set of proteins expressed in a given biological milieu such as cells, tissues, or fluids, has become the next holy grail of biomedical research. Although the genomes of over one hundred fifty species have been cataloged, a truly functional understanding of the difference between diseased and normal operating states of biological systems requires a correlation of the sequence of gene activity with the corresponding profile of expressed proteins. Gene transcription analysis is not sufficient to achieve this correlation, since many genes are expressed indirectly, e.g., through processing by the so-called spliceosome, regulated by so-called interference RNA (“RNAi”), or the expressed proteins are modified by post-translational factors. Thus, a catalog of proteins must be derived directly.
The two most discriminating techniques for separating component proteins from mixtures of proteins are the protein's isoelectric point and molecular mass: it is highly improbable that two different proteins could have exactly the same isoelectric point and the same molecular mass. These parameters have already been used in two-dimensional electrophoresis (“2-DE”), in which proteins in a mixture are separated first by isoelectric point followed by electrophoretic migration through a sodium dodecylsulfate (“SDS”) matrix. However, the use of SDS electrophoresis is not considered to be very efficient for the sort of large scale analysis necessary for proteomic or clinical applications.
Mass spectrometry is a much more powerful technique to determine not only mass, but protein identity by correlating a sample's fragmentation pattern with the fragmentation patterns of known proteins. One approach uses a 2-DE separation using a polyacrylamide gel plate and then each single separated spot is removed from the gel and analyzed by mass spectrometry. However, this is unpractical, because it is too laborious to catalog the thousands of proteins expressed in a typical proteome. A more practical approach would avoid separating proteins from a gel matrix.
Cretich, et al., (“Separation of Proteins in a Multicompartment Electrolyzer with Chambers Defined by a Bed of Gel Beads”, Electrophoresis 24 (2002) 577-581) describes the separation of proteins based on their isoelectric point using a matrix of cross-linked acrylamide and acrylamide monomers known to those of skill in the art under the trade name IMMOBILINE™ (available from Amersham Biosciences, Uppsala, Sweden). However, the mechanical characteristics of the matrix were less than satisfactory, which lead the authors to suggest the use of electrically inert porous beads. But such beads are not described.
Auto-buffering beads using mixtures of monomers has been described for the maintenance of pH at constant levels in a modifiable environment (e.g., vegetable growth). These beads are described in the literature (see, e.g., Righetti, P. G., Chiari, M. and Crippa, L.: “Macroreticulate Buffers: a Novel Approach to pH Control in Living Systems”, J. Biotechnol. 17 (1991) 169-176; Chiari, M., Pagani, L. and Righetti, P. G.: “Physico-Chemical Properties of Amphoteric, Isoelectric, Macroreticulate Buffers”, J. Biochem. Biophys. Methods 23 (1991) 115-130; Pompei, R., Lampis, G., Chiari, M. and Righetti, P. G.: “Culture of Eukaryotic Cells with Macroreticulate Buffers: Fermentation of Cellulolytic Fungi”, BioTechniques 11 (1991) 701-706). They contained, grafted onto the same, neutral polymer backbone, both the buffering ion and the counter ion needed for titrating the buffering ion around its pK value; so as to ensure maximum buffering capacity. Such beads, however, could only be used for buffering cell growth media, and the like. They could not be adopted as ion exchangers, since their very nature of containing both types of groups—both positively and negatively charged—would render the beads useless as protein exchangers.
Thus, a need remains for a medium for separating mixture components on the basis of their pI using a robust material. The present invention meet this and other needs.